Aflatoxin detoxification agent as well as preparation method and application thereof
A modified attapulgite-laccase composite addresses the inefficiencies of existing aflatoxin detoxification agents by simultaneously adsorbing and degrading aflatoxin, offering enhanced removal capacity and stability.
Patent Information
- Application Number
- CN202411539226.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2024-12-20
AI Technical Summary
Existing aflatoxin detoxification agents either require large dosages with poor adsorption efficiency or can only decompose aflatoxin but not adsorb it effectively, lacking simultaneous adsorption and degradation capabilities.
A modified attapulgite carrier is used to immobilize laccase, with a specific ratio and concentration, enhancing both adsorption and degradation of aflatoxin through a composite detoxification agent.
The modified attapulgite-laccase composite achieves improved aflatoxin removal with increased loading capacity and stability, maintaining high enzyme activity under long-term storage and high temperatures.
Smart Images
Figure CN119158537A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of aflatoxin detoxifying agents, and specifically relates to an aflatoxin detoxifying agent and a preparation method and application thereof. Background Art
[0002] Common aflatoxin detoxification agents are adsorption-type detoxification agents or degradation-type detoxification agents. The components in adsorption-type detoxification agents have the ability to adsorb aflatoxin, fix aflatoxin on its surface or inside by physical or chemical means, reduce its concentration and thus reduce absorption in the body. Microorganisms in degradation-type detoxification agents may produce secondary metabolites or secrete specific enzymes, which can decompose the toxic groups of aflatoxin and convert them into non-toxic or low-toxic degradation products.
[0003] However, the adsorption type detoxification agent has a large dosage, poor adsorption effect, and aflatoxin needs to be further treated after adsorption. The degradation type detoxification agent can decompose aflatoxin, but the above detoxification agents cannot achieve the same effects of adsorption and degradation at the same time. Summary of the invention
[0004] In order to solve the problems of high cost, large dosage and poor adsorption efficiency of detoxification agents in the prior art, the present invention provides an aflatoxin detoxification agent and a preparation method and application thereof.
[0005] To achieve the above objectives, the specific technical solutions of the present invention are as follows.
[0006] The first aspect of the present invention provides an aflatoxin detoxifying agent, wherein the aflatoxin detoxifying agent uses modified attapulgite as a carrier, and the laccase in the laccase solution is fixed on the modified attapulgite;
[0007] The usage ratio of the modified attapulgite to the laccase solution is 1 g: 2 mL to 6 mL; the concentration of the laccase solution is 2 U / mL to 20 U / mL;
[0008] The modified attapulgite is acid-modified attapulgite or organic-modified attapulgite.
[0009] The present invention uses modified attapulgite as a carrier, mixes a laccase solution with a concentration of 2U / mL to 20U / mL in a ratio of 1g:2mL to 6mL of the mass volume ratio of the modified attapulgite to the laccase solution, fixes the laccase on the modified attapulgite, utilizes the modified attapulgite to adsorb aflatoxin, and the laccase degrades the adsorbed aflatoxin, thereby obtaining a composite detoxification agent having both biological and physical effects, thereby effectively improving the removal effect of aflatoxin.
[0010] In another preferred embodiment, the specific preparation process of the acid-modified attapulgite is as follows:
[0011] The attapulgite is mixed with an acid solution and reacted at 25°C to 60°C to obtain an acid-modified attapulgite; the acid solution is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 1moL / mL to 5moL / mL; the mass volume ratio of the attapulgite to the hydrochloric acid solution is 10g to 20g: 1mL;
[0012] The specific preparation process of the organic modified attapulgite is as follows:
[0013] The acid-modified attapulgite is mixed with (3-aminopropyl)triethoxysilane, and stirred for reaction to obtain an organically modified attapulgite; the mass volume ratio of the acid-modified attapulgite to the (3-aminopropyl)triethoxysilane is 1 g to 3 g: 1 mL 。
[0014] In another preferred embodiment, the mesh size of the attapulgite is 200-600 meshes.
[0015] The second aspect of the present invention provides a method for preparing the aflatoxin detoxifying agent, comprising the following steps:
[0016] The modified attapulgite is mixed with the laccase solution to obtain a mixed solution, glutaraldehyde is added to the mixed solution, the pH is adjusted to 5-6, an immobilization reaction is carried out, the mixture is shaken at 30°C for 4 hours, and then centrifuged to separate the precipitate and the supernatant, and the precipitate is washed three times with a citrate buffer solution to obtain an aflatoxin detoxifying agent.
[0017] In another preferred embodiment, the mass percentage of glutaraldehyde in the mixed solution is 2.5% to 12.5%.
[0018] In another preferred embodiment, the immobilization time is 2 h to 8 h, and the temperature is 30° C. to 40° C.
[0019] In another preferred embodiment, the reagent used to adjust the pH value is citric acid-disodium hydrogen phosphate buffer or sodium hydroxide solution.
[0020] The third aspect of the present invention provides a method for degrading aflatoxin in peanut meal, wherein the aflatoxin detoxifying agent is mixed with feed to degrade the aflatoxin in the feed;
[0021] The mass ratio of the feed to the aflatoxin detoxifying agent is 15g:5mg-15mg.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] (1) The aflatoxin detoxifying agent of the present invention, the modified attapulgite can have a better adsorption effect on aflatoxin, and then the laccase fixed on the modified attapulgite is used to degrade the adsorbed aflatoxin. By controlling the concentration of the laccase solution to 2U / mL to 20U / mL, and the mass volume of the modified attapulgite and the laccase solution to 1g:2mL to 6mL, the loading capacity of the laccase can be effectively increased, thereby improving the degradation effect of aflatoxin. The aflatoxin detoxifying agent of the present invention can achieve physical and biological dual removal of aflatoxin, and the maximum removal amount of aflatoxin is 2μg / mg to 3μg / mg.
[0024] (2) In the process of preparing the aflatoxin detoxifying agent, the present invention can further increase the loading amount of laccase and the activity of laccase by controlling the reaction time, pH value and the concentration of the coupling agent, thereby improving the degradation effect of aflatoxin. In addition, the aflatoxin detoxifying agent in the present invention has good stability and still has good enzyme activity under long-term storage and high temperature. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a diagram showing the adsorption effect of attapulgite modified with different acidity on aflatoxin in the examples of the present invention.
[0026] Figure 2 This is a diagram showing the adsorption effect of attapulgite with different organic modifications on aflatoxin in the examples of the present invention.
[0027] Figure 3 This is a diagram showing the adsorption effect of different aflatoxin detoxifying agents modified by laccase on aflatoxin.
[0028] Figure 4 This is a graph showing the effect of different laccase loadings on the activity of laccase-modified aflatoxin detoxifying agent.
[0029] Figure 5 This is a graph showing the effect of different curing times on the activity of laccase-modified aflatoxin detoxifying agent.
[0030] Figure 6 This is a graph showing the effect of different curing temperatures on the activity of laccase-modified aflatoxin detoxifying agent.
[0031] Figure 7 This is a graph showing the effect of different pH values on the activity of laccase-modified aflatoxin detoxifying agent.
[0032] Figure 8 This is a graph showing the effect of glutaraldehyde on the activity of laccase-modified aflatoxin detoxifying agent.
[0033] Fig. 9 This is a scanning electron micrograph of ATP.
[0034] Fig.10 This is a scanning electron micrograph of HATP.
[0035] Fig.11 This is a scanning electron micrograph of AATP.
[0036] Fig.12 This is a scanning electron micrograph of L-AATP.
[0037] Fig.13 It is the specific surface area diagram of ATP, HATP, AATP, and L-AATP, where a is the porosity diagram.
[0038] Fig.14 These are the infrared spectra of ATP, HATP, AATP, and L-AATP.
[0039] Fig.15 These are the X-ray diffraction patterns of ATP, HATP, AATP, and L-AATP.
[0040] Fig.16 These are the results of aflatoxin detoxification tests of L-AATP, AATP and La at different times. DETAILED DESCRIPTION
[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0042] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.
[0043] The attapulgite was purchased from Dingbang Mineral Products Technology Co., Ltd.
[0044] Example 1
[0045] Optimization of Modified Attapulgite Conditions
[0046] 1. Optimization of acid modification conditions
[0047] 1) The attapulgite with mesh sizes of 200, 400 and 600 were mixed with hydrochloric acid with a concentration of 1 mol / mL, respectively, and reacted at room temperature for 16 h to obtain acid-modified attapulgite with different mesh sizes, which were recorded as HATP-1 group.
[0048] 2) The attapulgite with mesh sizes of 200, 400 and 600 were respectively mixed with hydrochloric acid with a concentration of 2 mol / mL and reacted at room temperature for 16 h to obtain acid-modified attapulgite with different mesh sizes, which were recorded as HATP-2 group.
[0049] 3) The attapulgite with mesh sizes of 200 mesh, 400 mesh and 600 mesh were mixed with 3 mol / mL hydrochloric acid, respectively, and reacted at 60° C. for 16 h to obtain acid-modified attapulgite with different mesh sizes, which were recorded as HATP-3 group.
[0050] 4) The attapulgite with mesh sizes of 200 mesh, 400 mesh and 600 mesh were mixed with hydrochloric acid with a concentration of 4 mol / mL, respectively, and reacted at 60° C. for 16 h to obtain acid-modified attapulgite with different mesh sizes, which were recorded as HATP-4 group.
[0051] 5) The attapulgite with mesh sizes of 200, 400 and 600 were respectively mixed with hydrochloric acid with a concentration of 5 mol / mL, and reacted at 60°C for 16 hours to obtain acid-modified attapulgite with different mesh sizes, which were recorded as HATP-5 group.
[0052] The acid-modified attapulgite in the HATP-1 group to the HATP-5 group in the above 1) to 5) and the unmodified attapulgite, denoted as ATP, were subjected to aflatoxin adsorption experiments. The results are as follows: Figure 1 As shown, from Figure 1 It can be seen from the results that when the mesh size of the attapulgite is 200 mesh and the hydrochloric acid concentration is 4 mol / mL, the acid-modified attapulgite has the best adsorption effect on aflatoxin.
[0053] 2. Optimization of organic modification conditions
[0054] 1) The acid-modified attapulgite with different mesh sizes in the HATP-4 group was mixed with 1 mL of (3-aminopropyl)triethoxysilane, and the mixture was stirred and reacted at room temperature for 16 h to obtain a modified attapulgite, which was recorded as AATP-1.
[0055] 2) The acid-modified attapulgite of different mesh sizes in the HATP-4 group was mixed with 1.5 mL of (3-aminopropyl)triethoxysilane, and the mixture was stirred and reacted at room temperature for 16 h to obtain modified attapulgite, which was recorded as AATP-2.
[0056] 3) The acid-modified attapulgite with different mesh sizes in the HATP-4 group was mixed with 2 mL of (3-aminopropyl)triethoxysilane, and the mixture was stirred and reacted at room temperature for 16 h to obtain modified attapulgite, which was recorded as AATP-3.
[0057] 4) The acid-modified attapulgite with different mesh sizes in the HATP-4 group was mixed with 2.5 mL of (3-aminopropyl)triethoxysilane, and the mixture was stirred and reacted at room temperature for 16 h to obtain modified attapulgite, which was recorded as AATP-4.
[0058] The organically modified attapulgite in the AATP-1 group to the AATP-4 group in the above 1) to 4) and the unmodified attapulgite, denoted as ATP, were subjected to aflatoxin adsorption experiments. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen from the results that when the mesh size of the attapulgite is 200 mesh and the dosage of (3-aminopropyl)triethoxysilane is 1.5 mL, the organically modified attapulgite has the best adsorption effect on aflatoxin.
[0059] Example 2
[0060] The concentration of the laccase solution in the preparation method of the aflatoxin detoxification agent is optimized. The acid-modified attapulgite obtained by setting the mesh number of the attapulgite to 200 meshes and the hydrochloric acid concentration to 4 mol / mL is recorded as HATP, and the organic-modified attapulgite obtained by mixing HATP with 1.5 mL of (3-aminopropyl) triethoxysilane is recorded as AATP. The specific process is as follows:
[0061] 1) 5 mg of ATP, AATP and HATP were mixed with 10 mL of laccase solution with a concentration of 2 U / mL, and the immobilization reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0062] 2) 5 mg of ATP, AATP and HATP were mixed with 10 mL of laccase solution with a concentration of 6 U / mL, and the immobilization reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0063] 3) 5 mg of ATP, AATP and HATP were mixed with 10 mL of 10 U / mL laccase solution, respectively, and the immobilization reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0064] 4) 5 mg of ATP, AATP and HATP were mixed with 10 mL of laccase solution with a concentration of 15 U / mL, and the immobilization reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0065] 6) 5 mg of ATP, AATP and HATP were mixed with 10 mL of 20 U / mL laccase solution, and the immobilization reaction was carried out for 12 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0066] The laccase loading and laccase activity of the different aflatoxin detoxifying agents obtained above were determined. Figure 3 As shown, from Figure 3It can be seen that when the concentration of the laccase solution is 15U / mL, the laccase loading and the relative activity of laccase are the highest, and the laccase loading and the relative activity of laccase of the organically modified aflatoxin detoxifier are better than those of the unmodified attapulgite and the acid-modified aflatoxin detoxifier.
[0067] Example 3
[0068] The optimization of the amount of laccase added in the preparation method of the aflatoxin detoxification agent comprises the following steps:
[0069] 1) 5 mg of ATP, AATP and HATP were mixed with 10 mL of laccase solution with a concentration of 15 U / mL, and the solidification reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0070] 1) 5 mg of ATP, AATP and HATP were mixed with 15 mL of laccase solution with a concentration of 15 U / mL, and the solidification reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0071] 2) 5 mg of ATP, AATP and HATP were mixed with 20 mL of laccase solution with a concentration of 15 U / mL, and the solidification reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0072] 3) 5 mg of ATP, AATP and HATP were mixed with 25 mL of laccase solution with a concentration of 15 U / mL, and the solidification reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0073] 4) 5 mg of ATP, AATP and HATP were mixed with 30 mL of laccase solution with a concentration of 15 U / mL, respectively, and the solidification reaction was carried out for 12 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0074] The laccase loading and laccase activity of the above-obtained aflatoxin-free agents were measured. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that when the addition amount of laccase is 25mL, the laccase loading and the relative activity of laccase are the highest, and the laccase loading and the relative activity of laccase of the organically modified aflatoxin detoxifier are better than those of the unmodified attapulgite and the acid-modified aflatoxin detoxifier.
[0075] Example 4
[0076] The optimization of the immobilization time in the preparation method of the aflatoxin detoxification agent comprises the following steps:
[0077] 1) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution for 1 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0078] 2) 5 mg of ATP, AATP and HATP were mixed with 25 mL of laccase solution with a concentration of 15 U / mL, respectively, and the solidification reaction was carried out for 2 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0079] 3) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and the solidification reaction was carried out for 4 hours to obtain L-ATP, L-AATP and L-HATP, respectively.
[0080] 4) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and the solidification reaction was carried out for 8 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0081] 5) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and the solidification reaction was carried out for 12 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0082] The laccase loading and laccase activity of the different aflatoxin detoxifying agents obtained above were determined. Figure 5 As shown, from Figure 5 It can be seen that when the immobilization time is 4h, the laccase loading and the relative activity of laccase are the highest, and the laccase loading and the relative activity of laccase of the organically modified aflatoxin detoxifier are better than those of the unmodified attapulgite.
[0083] Example 5
[0084] The optimization of the immobilization temperature in the preparation method of the aflatoxin detoxification agent comprises the following steps:
[0085] 1) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and solidified at 4° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0086] 2) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and solidified at 10° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0087] 3) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and solidified at 20° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0088] 4) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and solidified at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0089] 5) 5 mg of ATP, AATP and HATP in Example 1 were mixed with 25 mL of 15 U / mL laccase solution, respectively, and solidified at 40° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0090] The laccase loading and laccase activity of the different aflatoxin detoxifying agents obtained above were determined. Figure 6 As shown, from Figure 6 It can be seen that when the immobilization temperature is 30°C, the laccase loading and the relative activity of laccase are the highest, and the laccase loading and the relative activity of laccase of the organically modified aflatoxin detoxifier are better than those of the unmodified attapulgite.
[0091] Example 6
[0092] The optimization of pH value in the preparation method of aflatoxin detoxification agent comprises the following steps:
[0093] 1) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution, respectively, the pH was adjusted to 3, and the immobilization reaction was carried out at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0094] 2) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution, respectively, the pH was adjusted to 4, and the immobilization reaction was carried out at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0095] 3) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution, respectively, the pH was adjusted to 5, and the immobilization reaction was carried out at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0096] 4) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution, respectively, the pH was adjusted to 6, and the immobilization reaction was carried out at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0097] 5) 5 mg of ATP, AATP and HATP were mixed with 25 mL of 15 U / mL laccase solution, respectively, the pH was adjusted to 7, and the immobilization reaction was carried out at 30° C. for 4 h to obtain L-ATP, L-AATP and L-HATP, respectively.
[0098] The laccase loading and laccase activity of the different aflatoxin detoxifying agents obtained above were determined. Figure 7 As shown, from Figure 7 It can be seen that when pH is 5, the laccase loading and the relative activity of laccase are the highest, and the laccase loading and the relative activity of laccase of the organically modified aflatoxin detoxifying agent are better than those of the unmodified attapulgite and the acid-modified aflatoxin detoxifying agent.
[0099] Example 7
[0100] The optimization of the mass percentage of glutaric acid in the preparation method of the aflatoxin detoxification agent comprises the following steps:
[0101] 1) 5 mg of AATP was mixed with 25 mL of a laccase solution having a concentration of 15 U / mL and a pH of 5, and a curing reaction was performed at 30° C. for 4 h to obtain L-AATP.
[0102] 2) 5 mg of AATP was cross-linked with 2.5% glutaraldehyde for 2 h, washed and dried, and then mixed with 25 mL of laccase solution with a concentration of 15 U / mL and a pH of 5, and cured at 30° C. for 4 h to obtain L-AATP.
[0103] 3) 5 mg of AATP was cross-linked with 5% glutaraldehyde for 2 h, washed and dried, and then mixed with 25 mL of laccase solution with a concentration of 15 U / mL and a pH of 5, and cured at 30° C. for 4 h to obtain L-AATP.
[0104] 4) 5 mg of AATP was cross-linked with 7.5% glutaraldehyde for 2 h, washed and dried, and then mixed with 25 mL of laccase solution with a concentration of 15 U / mL and a pH of 5, and cured at 30° C. for 4 h to obtain L-AATP.
[0105] 5) 5 mg of AATP was cross-linked with 10% glutaraldehyde for 2 h, washed and dried, and then mixed with 25 mL of laccase solution with a concentration of 15 U / mL and a pH of 5, and cured at 30° C. for 4 h to obtain L-AATP.
[0106] The laccase loading and laccase activity of the aflatoxin detoxifying agent obtained above were measured. The results are as follows: Figure 8 As shown, from Figure 8 It can be seen that when the mass percentage of glutaraldehyde is 7.5%, the laccase loading and the relative activity of laccase are the highest.
[0107] From the results of Examples 2 to 7, it can be seen that the laccase loading and laccase relative activity of the aflatoxin detoxification agent obtained by using acid-modified attapulgite to fix laccase are lower than those of the aflatoxin detoxification agent obtained by using organic-modified attapulgite to fix laccase. The laccase loading and laccase relative activity of the aflatoxin detoxification agent obtained by using organic-modified attapulgite to fix laccase can reach up to 97.04 mg / g and the laccase relative activity can reach up to 98.01%. The optimal preparation conditions are a laccase concentration of 15 U / mL, a laccase solution dosage of 25 mL, an immobilization time of 4 h, a temperature of 30°C, a pH of 5, and a glutaraldehyde dosage of 7.5%.
[0108] In order to further illustrate the stability of the aflatoxin detoxifying agent, the aflatoxin detoxifying agent obtained by the optimal preparation conditions of the above-mentioned organic modified attapulgite fixed laccase was taken as an example, denoted as L-AATP, and the stability test was carried out with the free laccase denoted as La. The results showed that the enzyme activity of L-AATP remained above 90% of the initial enzyme activity after storage for 30 days, while the activity of La decreased to below 80% of the initial enzyme activity. Moreover, the high temperature resistance of L-AATP is significantly better than that of La. When the maximum temperature is 80°C, the activity of L-AATP still remains above 75% of the initial activity, and the activity of La decreases to below 50% of the initial activity. It can be explained that the aflatoxin detoxifying agent in the present invention can maintain a high enzyme activity for a long time and at a high temperature.
[0109] In order to further illustrate the structure of the modified attapulgite, the unmodified attapulgite was recorded as ATP, and the acid-modified attapulgite obtained under the optimal acid modification conditions, that is, the attapulgite mesh number was 200 mesh and the hydrochloric acid concentration was 4 moL / mL, was recorded as HATP; the modified attapulgite prepared by using the attapulgite mesh number of 200 mesh and the (3-aminopropyl)triethoxysilane dosage of 1.5 mL was recorded as AATP, and the organic modified attapulgite immobilized with laccase prepared under the optimal conditions was recorded as L-AATP, which were characterized by SEM, XRD, FT-IR and BET, respectively. The results are shown in Figures 9 to 15 shown.
[0110] From the scanning electron micrographs of ATP, HATP, AATP, and L-AATP, Figures 9 to 12 It can be seen that the unmodified attapulgite is a loose rod-shaped aggregate, and the acid modification and organic modification do not destroy the rod-shaped aggregate structure on the surface of the modified attapulgite.
[0111] from Fig.13 It can be seen that the specific surface area of ATP is 107.1053m 2 / g, total porosity 0.224641cm 3 / g; HATP specific surface area 174.9138m 2 / g, total pore size is 0.334195cm 3 / g; AATP specific surface area 56.3614m 2 / g, total porosity 0.223015cm 3 / g. The specific surface area of L-AATP is 60.5900m 2 / g, pore size is 0.205388cm 3 / g.
[0112] From the infrared spectra and X-ray diffraction patterns of ATP, HATP, AATP, and L-AATP, Fig.14 and Fig.15 It can be seen that the modification and immobilization did not change the crystal structure of the original ATP, and (3-aminopropyl)triethoxysilane was successfully grafted on the surface of ATP, and laccase was also successfully coupled to the organically modified attapulgite.
[0113] L-AATP, AATP and La were tested for aflatoxin detoxification. L-AATP, AATP and La were mixed with aflatoxin working solution at a ratio of 1 mg: 1 mL (m / v), placed in a shaker to react in a dark place, and shaken at 37°C in a dark place for 24 hours. The residual amount of aflatoxin B1 was measured by HPLC and recorded as AFB1. The results are as follows Fig.16 As shown, from Fig.16 It can be seen that the reaction reached equilibrium at 24 h, the detoxification capacity was L-AATP>AATP>La, and the detoxification rate of L-AATP was 98.55%.
[0114] Weigh 15 g of peanut meal with an aflatoxin content of 60 ng / mg, add aflatoxin detoxifying agent in a ratio of 1000:1 to 3000:1, mix thoroughly, then add 75 ml of simulated gastrointestinal fluid, shake the reaction for 24 hours, measure the toxin residue, and calculate the detoxification effect, as shown in Table 1.
[0115] Table 1 shows the detoxification test results of different mixing ratios
[0116]
[0117]
[0118] From the results in Table 1, it can be seen that the detoxification effect is better when the detoxification dosage of peanut meal and aflatoxin is 1500:1.
[0119] Different mixing times were explored, and the aflatoxin detoxification agent and peanut meal were mixed at a ratio of 1:1500 and then placed for different periods of time to measure the aflatoxin residues. The results are shown in Table 2. The aflatoxin content in the peanut meal was 60 ng / mg.
[0120] Table 2 Detoxification results of aflatoxin at different mixing times
[0121]
[0122] As can be seen from Table 2, the mixing time has little effect on the detoxification rate, which indicates that the detoxification agent of the present invention has good stability.
[0123] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0124] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. An aflatoxin detoxifying agent, It is characterized in that The aflatoxin detoxification agent uses modified attapulgite as a carrier, and fixes the laccase in the laccase solution on the modified attapulgite; The mass volume ratio of the modified attapulgite to the laccase solution is 1 g: 2 mL to 6 mL; the concentration of the laccase solution is 2 U / mL to 20 U / mL; The modified attapulgite is acid-modified attapulgite or organic-modified attapulgite.
2. The aflatoxin detoxifying agent according to claim 1, It is characterized in that The specific preparation process of the acid-modified attapulgite is as follows: The attapulgite is mixed with an acid solution and reacted at 25°C to 60°C to obtain an acid-modified attapulgite; the acid solution is a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 1moL / mL to 5moL / mL; the mass volume ratio of the attapulgite to the hydrochloric acid solution is 10g to 20g: 1mL; The specific preparation process of the organic modified attapulgite is as follows: The acid-modified attapulgite and (3-aminopropyl)triethoxysilane are mixed and stirred for reaction to obtain organically modified attapulgite; the mass volume ratio of the acid-modified attapulgite to the (3-aminopropyl)triethoxysilane is 1 g to 3 g: 1 mL.
3. The aflatoxin detoxifying agent according to claim 2, It is characterized in that The mesh number of the attapulgite is 200-600 meshes.
4. A method for preparing the aflatoxin detoxifying agent according to any one of claims 1 to 3, It is characterized in that The following steps are involved: The modified attapulgite is mixed with the laccase solution to obtain a mixed solution, glutaraldehyde is added to the mixed solution, the pH value is adjusted to 5-6 for immobilization reaction, and the precipitate is filtered to obtain the aflatoxin detoxification agent.
5. The method for preparing the aflatoxin detoxifying agent according to claim 4, It is characterized in that The mass percentage of the glutaraldehyde in the mixed solution is 2.5% to 12.5%.
6. The method for preparing the aflatoxin detoxifying agent according to claim 4, It is characterized in that The immobilization time is 2 hours to 8 hours, and the temperature is 30° C. to 40° C.
7. The method for preparing the aflatoxin detoxifying agent according to claim 4, It is characterized in that The reagent used to adjust the pH value is citric acid-disodium hydrogen phosphate buffer or sodium hydroxide solution.
8. A method for degrading aflatoxin in feed, It is characterized in that Mixing the aflatoxin detoxifying agent according to claim 1 with feed to degrade the aflatoxin in the feed; The mass ratio of the feed to the aflatoxin detoxifying agent is 15g:5mg-15mg.